Naturally occurring resistance-associated substitutions (RASs) can negatively impact the response to direct-acting antivirals (DAAs) agents-based therapies for hepatitis C virus (HCV) infection. Herein, we set out to characterize the RASs in the HCV1b genome from serum samples of DAA-na&iuml;ve patients in the context of the SINERGIE (South Italian Network for Rational Guidelines and International Epidemiology, 2014) project. We deep-sequenced the NS3/4A protease region of the viral population using the Ion Torrent Personal Genome Machine, and patient-specific majority rule consensus sequence summaries were constructed with a combination of freely available next generation sequencing data analysis software. We detected NS3/4A protease major and minor variants associated with resistance to boceprevir (V36L), telaprevir (V36L, I132V), simeprevir (V36L), and grazoprevir (V36L, V170I). Furthermore, we sequenced part of HCV NS5B polymerase using Sanger-sequencing and detected a natural RAS for dasabuvir (C316N). This mutation could be important for treatment strategies in cases of previous therapy failure.

ijms-17-01416-f001: RAxML phylogenetic trees were estimated using 24 hepatitis C virus (HCV) reference sequences (black) downloaded from Los Alamos HCV Sequence Database and 8 HCV isolates (red) in this study for NS3 (A) and NS5B (B) regions, respectively. The reliability of the phylogenetic clustering was evaluated using bootstrap analysis with 1000 replicates. Bootstrap support values are only shown for the HCV1b clades (yellow area). The scale bars at the bottom of the figure represent genetic distance.

Mentions:
The Versant HCV genotype 2.0 assay classified HCV isolates from all patients as HCV subtype 1b, except for one patient whose genotype was characterized as a mixed genotype 1b/4 (Table 1). In contrast, all samples were classified as genotype 1b by both the Oxford and COMET subtyping tools, and by phylogenetic analysis of the NS3 (Figure 1A) and NS5B (Figure 1B) regions.

ijms-17-01416-f001: RAxML phylogenetic trees were estimated using 24 hepatitis C virus (HCV) reference sequences (black) downloaded from Los Alamos HCV Sequence Database and 8 HCV isolates (red) in this study for NS3 (A) and NS5B (B) regions, respectively. The reliability of the phylogenetic clustering was evaluated using bootstrap analysis with 1000 replicates. Bootstrap support values are only shown for the HCV1b clades (yellow area). The scale bars at the bottom of the figure represent genetic distance.

Mentions:
The Versant HCV genotype 2.0 assay classified HCV isolates from all patients as HCV subtype 1b, except for one patient whose genotype was characterized as a mixed genotype 1b/4 (Table 1). In contrast, all samples were classified as genotype 1b by both the Oxford and COMET subtyping tools, and by phylogenetic analysis of the NS3 (Figure 1A) and NS5B (Figure 1B) regions.

Naturally occurring resistance-associated substitutions (RASs) can negatively impact the response to direct-acting antivirals (DAAs) agents-based therapies for hepatitis C virus (HCV) infection. Herein, we set out to characterize the RASs in the HCV1b genome from serum samples of DAA-na&iuml;ve patients in the context of the SINERGIE (South Italian Network for Rational Guidelines and International Epidemiology, 2014) project. We deep-sequenced the NS3/4A protease region of the viral population using the Ion Torrent Personal Genome Machine, and patient-specific majority rule consensus sequence summaries were constructed with a combination of freely available next generation sequencing data analysis software. We detected NS3/4A protease major and minor variants associated with resistance to boceprevir (V36L), telaprevir (V36L, I132V), simeprevir (V36L), and grazoprevir (V36L, V170I). Furthermore, we sequenced part of HCV NS5B polymerase using Sanger-sequencing and detected a natural RAS for dasabuvir (C316N). This mutation could be important for treatment strategies in cases of previous therapy failure.